EGF receptor (EGFR) overexpression occurs in over 50% of colorectal cancers (CRC) and is linked to metastasis and poor prognosis. Cetuximab, a monoclonal antibody against the EGFR, is approved by the FDA for the treatment of advanced CRC, but is effective in only 10% of CRC patients. We have shown that EGFR signaling plays an iterative role in intestinal neoplasia, acting at a post-initiation, establishment stage and again later during tumor progression. We recently found that Lrig1, a cell surface EGFR negative regulator that accelerates EGFR degradation, marks intestinal stem cells and that Lrig1 null mice develop intestinal tumors. These insights emerged from our generation and analysis of Lrig1-CreERT2 mice. Mig-6 is another EGFR negative regulator that is also expressed in the intestinal stem cell zone; it is a cytosolic protein that acts by blocking EGFR tyrosine kinase activity. Constitutive Mig-6 null mice develop rectal carcinoma but die at an early age because of severe rheumatological abnormalities. We have obtained mice with a conditional floxed allele of Mig-6. By crossing homozygous Lrig1-CreERT2 driver mice to conditional Mig-6 mice, we will selectively eliminate Lrig1 and Mig-6 in the intestine following injection of tamoxifen. Levels of both LRIG1 and MIG-6 are decreased in a number of human cancers, although immunohistochemical analysis has not been performed in CRC. We hypothesize that 1) targeted disruption of Lrig1 and Mig-6 in the intestinal progenitor compartment will lead to heightened and sustained Egfr signaling that predisposes to intestinal neoplasia and 2) their combined loss will result in more advanced tumors. We propose the following three specific aims to examine how loss of Lrig1 and Mig-6 contributes to intestinal neoplasia. Aim 1. Determine the mechanism of intestinal neoplasia in Lrig1 null mice. We will explore a two- compartment model of tumorigenesis based on dynamic crosstalk between the expanded Brunner's glands and the overlying epithelium. We will assess the importance of Egfr signaling in the tumor phenotype by crossing Lrig1 null mice to homozygous Wa2 mice, an Egfr hypomorph with markedly reduced Egfr activity. We predict these mice will not form tumors. Aim 2. Examine regulation of Lrig1 and Mig-6 in CRC cell lines in vitro and determine whether loss of both Lrig1 and Mig-6 in vivo enhances intestinal neoplasia. We will compare the efficacy of cetuximab in CRC cell lines that express (or not) these two EGFR negative regulators. We predict that restoring expression of these EGFR negative regulators will enhance the anti-tumor efficacy of cetuximab. By crossing homozygous Lrig1-CreERT2 mice to conditional Mig-6 mice, we will eliminate both Mig-6 and Lrig1 in the intestine upon tamoxifen-induced Cre activation. We predict there will be increased tumor burden in the colon and rectum. Aim 3. Determine whether LRIG1 and MIG-6 levels are decreased in human CRC and whether this decrease is clinically relevant. We will perform immunohistochemistry (IHC) for LRIG1, MIG-6 and total and phospho (p)-EGFR on two clinically and histologically annotated CRC tissue arrays: 1) primary human CRC and 2) matched primary carcinoma and liver metastasis. We predict that loss or reduced levels of LRIG1 and MIG-6 will correlate with increased levels of total and p-EGFR and that their combined loss will portend poor clinical outcomes. Analysis of these in vivo models, combined with in vitro cell culture systems and clinical samples, will provide new insights into pathogenesis of intestinal neoplasia and may lead to novel therapeutic strategies for CRC.